results of the european clostridium difficile survey...
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National Institute
for Public Health
and the Environment
Results of the European Clostridium difficile survey (ECDIS)
Martijn Bauer, MD; on behalf of the ECDIS Study Group and local coordinators
National Institute
for Public Health
and the Environment
Methods
November 2008, follow-up February 2009
Patients >2 years suspected of CDI or inpatients developing diarrhoea after ≥3 days of admission
CDI case definition: compatible clinical picture and positive stool test for C. difficile toxin
Of every first 10 patients per hospital:
questionnaires on inclusion and 3 months follow-up
stools cultured locally
isolates sent to Leiden University Medical Centre for PCR-ribotyping and testing for presence of toxin genes
National Institute
for Public Health
and the Environment
Analysis
Incidence rates of healthcare-associated CDI: (number of CDI cases x proportion of healthcare-associated CDI)/ patients-days or admissions
Odds ratios of patient and pathogen characteristics and outcome parameters (i.e., severe CDI and recurrence)
Relevant variables analyzed by logistic regression
National Institute
for Public Health
and the Environment
Healthcare-associated CDI (n/ 10,000 patient-days)
[0 – 2
[2 – 4
[4 – 6
[6 – 8
[8 – 10
[10 – 20
National Institute
for Public Health
and the Environment
Patient characteristics
509 included, 484 in follow-up
80% healthcare-associated
Age median 71 (IQR 56 – 81) yr
44% severe comorbidity, 50% immunocompromised
16% episodes of CDI in previous 8 weeks
79% antibiotics in previous month, 92% in previous 3 months
28% diarrhoea > 1 week
4% ileus
29% last leukocyte count ≥ 15 ∙ 109/L
National Institute
for Public Health
and the Environment
Follow-up after 3 months
7% ICU admissions
23% CDI contributive or primary cause
0.7% colectomies for CDI
22% died
40% CDI contributive or primary cause
18% recurrent CDI
National Institute
for Public Health
and the Environment
Determinants of severe CDI Univariate Multivariate
Characteristic OR 95%CI OR 95%CI
Age ≥ 65 years
4.87 1.88 – 12.63 3.44 1.12 – 10.52
Healthcare-associated
3.29 0.99 – 10.90
Severe comorbidity 1.17 0.61 – 2.23
Heart disease 1.52 0.60 – 3.85
Pulmonary disease
2.52 1.16 – 5.50
Antibiotics during previous month:
aminopenicillin + βL inh. 2.05 1.01 – 4.14
3rd or 4th generation fluoroquinolone
2.85 1.08 – 7.55
macrolide
2.59 0.91 – 7.36
Episodes of CDI in previous 8 weeks
0.84 0.31 – 2.24
PCR-ribotype:
027 4.18 1.03 – 17.05 5.56 1.29 – 23.92
015 5.78 1.59 – 20.95 9.06 2.31 – 35.47
018 7.10 2.53 – 19.94 7.20 2.45 – 21.14
National Institute
for Public Health
and the Environment
Determinants of recurrent CDI
Univariate Multivariate
Characteristic OR 95%CI OR 95%CI
Age ≥ 65 years
1.88 1.11 – 3.17 1.98 1.10 – 3.59
Healthcare-associated
1.95 0.96 – 3.93
Severe comorbidity
1.32 0.81 – 2.17
Antibiotics during previous month:
antipseudomonal penicillin + βL inh. 1.74 0.81 – 3.75
ceftazidime 2.12 1.19 – 3.78 2.22 1.16 – 4.26
glycopeptide
1.92 0.85 – 4.35
Episodes of CDI in previous 8 weeks 2.34 1.27 – 4.30 2.75 1.46 – 5.19
National Institute
for Public Health
and the Environment
Conclusions – surveillance Nov 2008
• The incidence of CDI varied widely in Europe
• Many PCR-ribotypes, in particular 014, 001 and 078
• Most cases healthcare-associated
• The classical risks old age, comorbidity and antibiotic use
• During follow-up, 22% of patients died (40% CDI contributive)
• Severe disease in elderly, PCR-ribotypes 015, 018 and 027
• Recurrent disease in elderly, ceftazidime use and number of prior episodes of CDI
• Clinical characteristics of CDI were not strongly correlated with a complicated course or recurrence of disease
National Institute
for Public Health
and the Environment
Limitations of study method
• Patients/ samples representative for whole country?
- maximum of 10 patients per hospital
- selection of hospitals
• Local toxin tests, culture methods and data retrieval varied
• Cases defined by toxin test, not culture
• Distribution across Europe: higher incidence or higher awareness?
PCR ribotypes in The Netherlands
0
50
100
150
200
250
300
2nd 3rd 4th 1st 2nd 3rd 4th 1st 2nd 3rd 4th 1st 2nd 3rd 4th 1st 2nd
2005 2006 2007 2008 2009
Quarter of a year
Nu
mb
er
of
iso
late
s Other
type 002
type 014
type 001
type 078
type 027
Emerging Clostridium difficile 078
Characteristics of the strain – tcdA and tcdB Positive – tcdC 39 bp deletion – tcdC mutation at 184, stopcodon – ermB Negative – Binary toxin Positive
Genotyping of the strain – PCR-ribotyping 078 – Toxinotyping V
– Further subtyping MLVA?
Clinical - Attributable mortality within 30 days: 3.8% - Complications: 9.6% - Relapse rate: 15.8% - Severe diarrhoea as 027, but affects younger patients
The Netherlands, Northern Ireland US: third most common prevalent type in CA-CDI European study 2005: 11th type France: 3,25% in 2006 to 11% in 2007
Two herds with outbreaks of diarrhoea in piglets (1 year)
Yellow to orange watery diarrhoea
High morbidity (80%), low mortality (12%), growth rates were affected
Periparturient medication of sows with trimethoprim-sulfadiazin, vaccination and use of amoxicilline:
Exsudative fibrino-haemorragic colitis of colon, but no necrotic lesions in mucosa of small intestine (C. perfringens). Mesocolonic oedema.
Cultures for C. perfringens negative.
No Isospora suis or rotavirus.
MLVA type 078
Clonal complex with Human and pig isolates
MLVA of 65 Clostridium difficile Type 078 isolates: 54 human isolates and 11 porcine isolates.
CLOSTRIDIUM DIFFICILE IN A FARROWING PEN
Hopman, N.E.M., Keessen, E.C., Harmanus, C, van Leengoed, L.A.G.M., Kuijper, E,
Lipman, L.J.A.
Dutch pig-breeding farm with 200 sows
All sampled 72 newborn piglets, irrespective of the presence of diarrhoea, acquired C. difficile 078 within two days after birth. Within this herd, just one ribotype, CD ribotype 078, was isolated from neonatal piglets, sows and from the environment (floor, air) of the piglets.
None of the 38 piglets born by caesarean section became positive for the presence of CD 078
Relatedness of human and animal Clostridium difficile PCR Ribotype 078 isolates (collaboration with Mark Wilcox, Leeds)
101 human isolates 44 Northern Ireland
20 other parts UK
3 Ireland
34 The Netherlands
56 porcine isolates 11 different pigfarms in 2006-2009
0
5
10
15
20
25
0,047 0,064 0,094 0,125 0,19 0,5 8 12 16 24 32 48 64
MIC (mg/ul)
Nu
mb
er
of
iso
late
s
Porcine
Human
74% of human type 078 strains, 27% porcine type 078 strains were resistant to tetracycline (MIC≥ 8 mg/l); p<0.05. All tetracycline resitant strains had Tn916-like transposon
>
23 CC (STRD ≤ 2); 5 CC human and animal isolates, 5 porcine, 13 human (6 specific region) 12 CC only tetracycline resistant isolates, 3 tetra susceptible, 8 mixed
Supporting capacity building for
surveillance of Clostridium difficile infections at European level
(2010-2013)
Tenderer: Ed J. Kuijper, Department of Medical Microbiology, Leiden University Medical Centre, Leiden, the Netherlands ECDC: Carl Suetens Investigator: drs. Marjolein Hensgens, LUMC/ RIVM, The Netherlands Manager: Walter Zuijderduin, LUMC, Leiden Website: www.ecdisnet.eu
Supporting capacity building for surveillance
of Clostridium difficile infections at European level
To enhance the laboratory capacity for
detection and surveillance of Clostridium difficile in European Member States (MS), Norway, Iceland and Liechtenstein.
To build up and maintain a European ribotyping nomenclature reference database for Clostridium difficile.
To develop a enhanced CDI surveillance protocol.
Beneficiary
Number
Beneficiary name Resonsible
coordinator
Country Leader
of WP
Participating
in WP
1
Leiden University Medical
Center, Leiden
E.J.Kuijper
NL
1
2,3,4
2
Leeds Teaching
Hospitals NHS Trust, &
Health Protection Agency
M.H.Wilcox
1
2,3,4
3
Center for Infectious
Diseases Control (Cib),
RIVM, Bilthoven
D.W
Notermans
NL
2
3,4
4
Anaerobe Reference
Laboratory, Cardiff, Wales
V. Hall
3
2,,4
5
Charité -
Universitätsmedizin Berlin
P. Gastmeier
Germany
4
2,3
Coordinators
Supporting capacity building for surveillance of Clostridium difficile infections at European level:
Other participants
National Public Health Institute, Helsinki (A. Virolaine , Outi Lyytikäinen )
University of Szeged, Szeged (E. Nagy)
National Institute of Health (ISS), Rome (P. Mastrantonio)
National Reference centre for HAI, Sofia (Rossitza Vatcheva-Dobrevska and K. Ivanova)
AGES-Institut für medizinische Mikrobiologie und Hygiene, Vienna (A. Indra)
University College Dublin and Health Protection Surveillance Centre (HPSC), Dublin (L. Kyne and F. Fitzpatrick)
Institut de Veille Sanitaire, Saint-Maurice Cedex (F. Barbut)
Health Protection Scotland, Glasgow (Camilla Wiuff)
Department of Epidemiology, Swedish Institute for Infectious Disease Control (Johan Struwe)
Work packages Work package Coordinators Time period
(1) Project Coordination Mark Wilcox and Ed Kuijper (Leeds and Leiden)
0-36 months
(2) Enhancing laboratory capacity for CDI detection in EU Member States.
Daan Notermans (RIVM, The Netherlands)
0-24 months
(3) Establishing a European ribotyping nomenclature reference database for Clostridium difficile in close collaboration with ECDC (TESSy).
Val Hall (Cardiff, Wales) 4-24 months
(4) To develop a European enhanced CDI surveillance protocol
Petra Gastmeier (Charité, Berlin)
0-24 months
Perform a feasibility study by implementing the protocol in at least 6 Member States
Petra and others 24-36 months
Work package 1; Project Coordination
Work package leaders: dr. Ed Kuijper (Leiden) and
prof. Mark Wilcox (Leeds)
Objective 1. Set up a project coordination group and a
network of representatives from each EU Member State, EU-MS, Norway, Iceland and Liechtenstein and candidate countrie
Objective 2. Communication between the consortium members and TESSY at ECDC.
Objective 3. Budgetary control.
Objective 4. Consortium reporting to the ECDC
Work package 2: Enhancing laboratory capacity for
CDI detection in EU Member States.
Work package leader: dr. Daan Notermans, CIb, RIVM, Bilthoven, The Netherlands.
Objective 1. Set up a network of CDI-reference labs
Objective 2. Perform an assessment of MS primary diagnostic laboratory capacity for Clostridium difficile and for typing capacity (ribotyping of CD isolates) and the need for training.
Objective 3. A proposal for standard operating procedures (SOPs) for the routine culture of Clostridium difficile isolates
Objective 4. A training module will be designed for culturing C. difficile and a re-assessment will be performed after implementation of the training module
Web based questionniare (Dr. Daan Notermans, RIVM, The Netherlands)
National coordinators of 32 countries were requested to select at random 10% of all laboratories to participate in a questionnairre on laboratory diagnostics
Minimum of 3 laboratories
31 coordinators replied
12/30 (38%) national guidelines to test for CDI
22/30 (71%) of the countries had a laboratory capable to type C. difficile
14/27 (52%) had “national reference laboratories” officially funded
20/22 laboratories performed PCR ribotyping
48 and 58% responded that training for culturing and typing was needed
Work package 3; Establishing a European ribotyping
nomenclature reference database for Clostridium difficile in close collaboration with ECDC (TESSy).
Work package leader: dr.Val Hall, ARU, Cardiff, UK.
Objective 1. Build up and maintain a ribotyping nomenclature reference database for Clostridium difficile.
Objective 2. Provide free of charge service to MS reference laboratories for sharing C. difficile reference strains.
Objective 3. Provide a written document on SOPs and propose a guideline for the ribotyping of Clostridium difficile isolates in EU
Objective 4. Provide External Quality Assesment (EQA) for national reference laboratories in the MS for ribotyping and assessment of antimicrobial resistance of C. difficile strains (yearly or 6-monthly
ARU collection of >15,000 C. difficile isolates
345 distinct ribotypes recognised
>1000 isolates of types 001, 027 & 106.
100-1000 isolates of 13 ribotypes.
11-100 isolates of 53 ribotypes.
<5 isolates of 226 ribotypes.
Most common types are in the ECDC/Leeds collection.
Establishment of a European ribotyping nomenclature reference database for C. difficile
PCR-ribotyping agarose gel method
1. Extract DNA from pure culture (<24h old) in 5% Chelex-100 resin. Heat at 100oC 12min.
2. Centrifuge, use supernate as template.
3. Amplify with O’Neill 16S – 23S primers.
Establishment of a European ribotyping nomenclature reference database for C. difficile
PCR-ribotyping agarose gel method
4. Concentrate amplicons at 75oC for ~45min.
5. Separate amplicons in Metaphor agarose gel (3%) with 100-1000bp ladder, 3h @ 60mA.
6. Capture image. Save as .tif file.
7. Use GelCompar / Bionumerics to compare band patterns with library of known ribotypes.
Establishment of a European ribotyping nomenclature reference database for C. difficile
Agarose- vs. capillary-gel methods
Agarose gel method
Only basic equipment needed
Proven technology
Database of 345 types established
Database not easily shared
Analysis is labour-intensive
Less practical for large numbers of isolates
Capillary gel method
High cost equipment
Evaluations in progress
Database to be constructed
Practical for inter-lab use
Less subjective analysis
Larger throughput possible
Establishment of a European ribotyping nomenclature reference database for C. difficile
Proposed network of typing labs
Regional lab 1
Regional lab 2 Regional
lab 5
National Ref lab A
Regional lab 4
Regional lab 3
National Ref lab B
Anaerobe Ref Unit, Cardiff, UK
Un
reco
gnis
ed
rib
oty
pe
Re
po
rt
New type to database
Establishment of a European ribotyping nomenclature reference database for C. difficile
Regional lab 7
Regional lab 6
CDC/PHAC/LUMC/Leeds C. difficile Typing Study
Dr. Duncan McCannel (CDC) Dr. Michael Mulvey (PHAC) Dr. Ed Kuijper (Leiden) Prof. Mark Wilcox (Leeds)
Aims
Compare PFGE with PCR ribotyping on a selected number of well defined C. difficile strains
Characterization of international set of reference C. difficile strains
Optimization of protocol for capillary gel electrophoresis PCR ribotyping
PFGE and PCR ribotyping
Leeds/Leiden collection (70 most frequently found isolates in Europe)
CDC: PFGE (SmaI, EagI, MluI), PCR (cdtB, lok1/3, tcdC), PCR-Ribotyping (CGE+agarose)
PHAC: PFGE (SmaI), PCR (tcdA, tcdB, tcdC, cdtB, tpi), PCR-Ribotyping (agarose)
Results; too many discrepancies and unclear nomenclature of PFGE
Results Leeds/Leiden collection
• Agreement of genetical characterization of Leeds/Leiden strains with exceptions of Types 078 and 126. Subtypes of 019 and 027?
• Disagreement of phenotypical characterization of toxin production A and B with presence of TcdA and TcdB
• Pilot (n=50) CE-PCR ribotyping using home made protocols: good agreement
Capillary gel electrophoresis PCR
ribotyping
Standardization of the protocol nearly achieved
Interlaboratory exchangeable files
Import in Bionumerics deserves more attention
Plans
New protocol of CE-PCR ribotyping is currently completed
Val Hall: validated 70 reference strains
Further expansion of database by Leeds, LUMC and Wales
Open library accesable
Work package 4: To develop a European enhanced CDI surveillance protocol
Work package leader: Prof. Dr. Petra Gastmeier, Charité - Universitätsmedizin Berlin, Germany.
Objective 1: Review methods and data of existing national CDI surveillance protocoll
Objective 2: Call an expert meeting to develop a European enhanced CDI surveillance protocol with case based epidemiological and microbiological (typing) data for infections.
Objective 3: Perform a feasibility study by implementing the protocol in at least 6 Member States (3 with high experience and 3 with no prior experience).
Objective 4: Presentation and agreement of the enhanced protocol during the annual Clostridium difficile network meeting
Components and interdependencies
RIVM: Centre for Infectious Diseases Control (Cib), RIVM, Bilthoven Berlin: Charité - Universitätsmedizin Berlin Cardiff: Anaerobe Reference Laboratory, University Hospital of Wales Leiden: Leiden University Medical Center, Leiden Leeds: Leeds Teaching Hospitals NHS Trust, Univ. of Leeds & Health Protection Agency